Bacteria that have developed resistance to conventional antibiotics are an increasing public health concern, and antimicrobial peptides (AMPs) represent a potential alternative to combat these bacteria. One intriguing family of AMPs is the histone-derived antimicrobial peptides (HDAPs). HDAPs have been isolated from natural sources, and the mechanisms for many of these peptides have been determined experimentally, such as in work performed during our previously funded AREA grant. Other work in our lab has designed a series of novel HDAPs based on histone crystal structures. However, relatively little effort has focused on the rational optimization of HDAPs to engineer more active peptides. The research in this proposal aims to address this gap by considering a systematic approach for the rational design of HDAPs. In this proposed design, we will focus on the role of three factors: the Arg composition of basic amino acids, the minimal length of active peptides and the creation of hybrid peptides. We will use a combination of bacterial assays, confocal microscopy, spectroscopic measurements, and molecular dynamics simulations to probe how each of these factors influences peptide activity on a molecular level. Although these factors are known to influence the activity of other AMPs, the structure-function relationships leading to these effects are not well understood. Thus, we expect that results from this proposed work can be generalized to give insight into the how these factors influence the activity of other AMPs. HDAPs are an ideal family of peptides to use for this purpose since they operate through different mechanisms, with some permeabilizing membranes and others translocating into cells and interacting with intracellular components. After elucidating trends for these three characteristics in HDAPs, we will combine this information to create a novel series of HDAPs that optimize the individual factors for peptide activity. We can thus validate whether this is a useful approach to peptide design in HDAPs that could be applied to other peptide systems. Together, the insight from these studies will both promote the development of HDAPs as potential therapeutic agents and provide insights into HDAP structure-function relationships that potentially can be generalized to other families of cationic AMPs. In addition to these scientific goals, this proposed research also has a strong emphasis on training as the work will be carried out by several undergraduate researchers and a few recent graduates at Wellesley College, an undergraduate women's college.